Hi everyone.. A brief intro first
I am trying to do one assay with my peptide conjugated with GFP and the control is GFP. I purified both proteins using Ni-NTA as they possess His-TAG. The purity is more than 95% on SDS gel. I then dialyze the protein against PBS and concentrate using Amicon filter.
So the problem is when I add the proteins on my cells in a 96 well plate for MTT assay, after 48 hours I see bacterial contamination in all my wells in test sample (peptide-GFP), control (GFP) and wells containing same vol of PBS.
The volume of concentrated protein is quite low so how should I filter or sterilize my protein it to prevent bacterial contamination? Please advice if anyone has done something similar....
Size: peptide-GFP= 30.7KDa, GFP= 27.6KDa
I did some similar experiments with liofilized rec proteins. They were dissolved in sterile PBS. For GFP, it likely should not work (ie, liofilization).
From a certain point you should work aseptically. It means a simple, extra clarifying your Amicon instruments with aseptic alcohol. You can work on your bench. Before concentration, filter your protein solution. No need a hood at first try but use gloves and use sterile tips and tubes throughout the procedure (concentrating).
Hello
i agree with Attila Lehotzky , also use new medium and reagents or check medium before plating
http://www.nbsbio.co.uk/downloads/PI_MTT-Stable-Solution.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4629458/.
http://home.sandiego.edu/~josephprovost/MTT%20Proliferation%20Assay%20Protocol.pdf
https://www.atcc.org/~/media/DA5285A1F52C414E864C966FD78C9A79.ashx
https://resources.rndsystems.com/pdfs/datasheets/ta5355.pdf
http://www.ncbi.nlm.nih.gov/pubmed/18345591
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